PROJECT SUMMARY/ABSTRACT Alzheimer's Disease (AD) is the most common cause of dementia, affecting 1 in 10 over the age of 65 in the United States. AD is debilitating and fatal, and no effective treatments are available. Dysregulation of autophagy, the process of transporting cellular waste to the endo-lysosomal system for degradation, has been proposed as a mechanism involved in AD and many other neurodegenerative diseases. Autophagy can be down-regulated by both normal aging and mutation of proteins with autophagic functions. The greatest genetic risk factor for late onset AD is APOE. I hypothesize that APOE functions as a selective autophagic receptor protein, and that the disease-associated APOE4 isoform dysregulates autophagy, contributing to disease. APOE4 impairs autophagy in mouse astrocytes, reduces autophagy gene transcription in a human glioblastoma cell line, and results in dramatic endosomal enlargement in the early stages of AD. APOE transports the majority of the brain's lipids, and lipid accumulation is one of the pathological hallmarks of AD, suggesting that lipid clearance may be impaired. It has not yet been tested whether APOE may impact autophagic function directly as part of the autophagy machinery. APOE has several properties of established autophagy receptors, including localization in acidic compartments and oligomerization. It was recently shown that APOE interacts with mammalian Atg8s during autophagy, a key characteristic of autophagy receptors. Protein sequence alignment data suggests that APOE may have some similarity to the yeast autophagy protein Atg39, a recently discovered receptor for the autophagy of lipids and the endoplasmic reticulum. The Aims of this proposal are as follows. Aim 1: Assess APOE interactions with the autophagy machinery. I will investigate APOE's function as an autophagic receptor protein in part by defining its interactions with autophagy machinery proteins. Aim 2: Examine accumulation of autophagic substrates upon APOE knockdown. Here, I will determine whether APOE is required for selective autophagy of lipids and endoplasmic reticulum. Aim 3: Assess impact of APOE isoforms on autophagic degradation. For this aim, I will assess whether the disease-causing APOE4 allele can impact autophagic turnover of APOE, autophagosome formation, and accumulation of autophagic substrates. These aims will begin to determine whether APOE has a novel function as an autophagic cargo receptor. Investigating the functions of APOE in autophagy, and whether the APOE4 isoform impairs or enhances those functions, may offer insight into the types of autophagy modifying therapies that could benefit millions of patients with AD.